PCR and RT-PCR are still experimental techniques, ISSN 2753-8176 (online), DOI:10.13140/RG.2.2.12112.17920

PCR and RT-PCR are still experimental techniques Ana Pedro1 1Gwyntwr1386 Healthcare CIC, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK 1. Corresponding author: anapedrolaboratories@gmail.com The polymerase chain reaction (PCR) is a technique currently used to rapidly amplify a very small sample of a complete or partial specific DNA sequence, which is exponentially amplified in a series of cycles of temperature changes to a large enough amount of DNA to be studied in detail. PCR was invented in 1983 by the American biochemist Kary Mullis (1). PCR uses short single strand DNA fragments (oligonucleotides) which are complementary sequences to the target DNA region in study named primers, a DNA polymerase and deoxynucleoside triphosphates (dNTPs, nucleotides containing triphosphate groups), which are the unit blocks from which the DNA polymerase synthesizes a new DNA strand. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature ( nucleic acid denaturation). In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to assemble a new DNA strand from free dNTPs. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified (1). The majority of the PCR methods amplify DNA fragments of between 0.1 and 10 kbp in size, however some techniques allow for amplification of fragments up to 40 kbp (2). The availability of substrates in the reaction limits the amount of amplified product, which becomes limiting as the reaction progresses (3). A variant of the PCR technique, reverse transcription polymerase chain reaction (RT-PCR) is a method combining reverse transcription of RNA into DNA (in this context named complementary DNA, cDNA) and the amplification of specific cDNA regions using PCR (4). In this case, the RNA template is converted into cDNA using a reverse transcriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. However, in most eukaryotic genes, coding regions (exons) are interrupted by non-coding regions (introns).During transcription, the entire gene is copied into a pre-mRNA, which includes exons and introns. During the process of RNA splicing, introns are removed and exons joined to form a contiguous coding sequence. Therefore, how it will be known if is being reversibly transcribing a pre-mRNA or a mature RNA sequence? And if it was reversibly transcribed a mature RNA sequence, how it can be known which was the original gene DNA sequence? Moreover, if all of the RNA in a cell is made by DNA transcription, therefore how does supposedly “independent viruses” can contain just RNA? And RNA is quite prone to a very quick degradation, how it can be assured, RNA will be in good conditions to be reversibly transcribed accurately? In addition, the behaviour of thermostable RTs remain poorly understood. RTs have a high error rate when transcribing RNA into DNA because, unlike most DNA polymerases, they have no proofreading ability allowing mutations to accumulate (5,6). Also, PCR and RT-PCR simply amplify genetic material present in a sample and there is no way to know if this genetic material is free or associated with any type of particles or vesicles or microorganisms unless an In situ hybridization technique is employed. By other side, gene regulation poorly understood and, therefore is not possible to know how it is related with disease processes whether an infectious or genetic or other type of disease (7). Cell differentiation in tissues and organs is dependent on the appropriate temporal and spatial control of gene expression within the many cells which make up the organism. Much is understood about how individual gene regulatory elements function, however many questions remain about how they interact to maintain correct regulation globally throughout the genome (8). In conclusion, because gene regulation is still poorly understood and these techniques still present major drawbacks their results should be interpreted with caution and they should remain as still experimental techniques and not as standardized diagnostic methods. References: 1. Mulis K et al.(1994). The Polimerase Chain Reaction. Springer Science+Business media New York 2. Cheng S, Fockler C, Barnes WM, Higuchi R (June 1994). "Effective amplification of long targets from cloned inserts and human genomic DNA". Proceedings of the National Academy of Sciences of the United States of America. 91 (12): 5695–99. Bibcode:1994PNAS...91.5695C. doi:10.1073/pnas.91.12.5695. PMC 44063. PMID 8202550. 3. Carr AC, Moore SD (2012). Lucia A (ed.). "Robust quantification of polymerase chain reactions using global fitting".PLOS ONE.7 (5): e37640. Bibcode:2012PLoSO...737640C.doi:10.1371/journal.pone.0037640. PMC 3365123. PMID 22701526. 4.Freeman WM, Walker SJ,Vrana KE (January 1999)."Quantitative RT-PCR: pitfalls and potential".BioTechniques.26 (1): 112–22, 124–5. doi:10.2144/99261rv01.PMID 9894600. 5. Bbenek K, Kunkel AT (1993). "The fidelity of retroviral reverse transcriptases". In Skalka MA, Goff PS (eds.). Reverse transcriptase. New York: Cold Spring Harbor Laboratory Press. p. 85. ISBN 978-0-87969-382-4. 6. "Promega kit instruction manual" (PDF). 1999. Archived from the original (PDF) on 2006-11-21. 7. Sahu et al (2022) Sequence determinants of human gene regulatory elements. Nature Genetics.54,283–294 8. Atkinson TJ, Halfon MS (2014) Regulation of gene expression inthe genomic context. Computational and Structural Biotechnology Journal. 9 (13): e201401001. doi: http:// dx.doi.orgl 10.59361csbj.20140 100 1